Squamous cell carcinoma (SCC) of the oral cavity and pharynx affect over 30,000 Americans each year. The 5-year survival for oral cancers is a poor 53% in whites, but is a staggeringly low 31% in blacks. Thus elucidation of factors responsible for this low mortality rate in African-Americans is important. Carcinomas probably develop as accumulations of genetic alterations which either activate proto-oncogenes or inactivate tumor suppressor genes (TSG). The oral cavity is an excellent anatomic site to study the steps in carcinogenesis. Some non-cancerous lesions of oral epithelium progress to squamous cell carcinomas over time. The diagnoses of these lesions range from acanthosis and/or keratosis to carcinoma-in- situ. Molecular analysis of possible genetic alterations in these pre- cancerous tissues should determine the stage at which a particular mutation occurs. Further, differences in genetic alterations of TSGs in oral SCCs may partially explain racial oral cancer mortality rate differences. Elucidation of the genetic mechanism of oral SCC tumor progression may ultimately reduce the high mortality rate in blacks. Improved tumor screening and treatment planning may be achieved through tests assessing TSGs. We will extend our earlier studies that showed the p53 TSG is inactivated in oral SCC by examining oral acanthotic, keratotic, dysplastic and carcinoma-in-situ oral epithelial cells. We will also investigate inactivation of the retinoblastoma (Rb) gene and adenomatous polyposis coli (APC) TSGs in oral carcinogenesis. Inactivations of these two TSGs have been found in several human tumors including cancer of the colon, lung, breast and esophagus. Precancerous or true cancer cells will be separated from normal cells by micro-dissection of fixed biopsies of oral epithelium. Fresh frozen tumor tissues will also be microdissected and utilized. DNA will be extracted from the malignant, "premalignant" and normal oral epithelial cells. Sections of the p53, APC, and Rb genes containing known DNA polymorphisms will be amplified using PCR. The amplified DNA will be digested with restriction enzymes that cut at these polymorphisms. Alterations of the tumor suppressor genes will be detected by the loss of one allele in those individuals who are heterozygous for the site in their normal cells. The use of multiple polymorphic sites within each gene will make most patients informative at these loci. Our previous studies revealed loss of heterozygosity (LOH) in p53 in 10 oral cancers from 14 heterozygous individuals. This project will further elucidate the role of tumor suppressor genes in oral cancer and begin to provide data for the development of a model of oral carcinogenesis. A collaboration between the University of Maryland at Baltimore, VCU and Howard University will allow the collection of sufficient tumor specimens from minority and female patients to also address the question of racial and gender differences in TSG inactivation. Inter-institutional collaboration will also extend to the engagement of a Howard University faculty member in the molecular analyses to be performed at Maryland.